The present invention relates to autoradiography compositions and methods. More particularly, the invention relates to methods and compositions useful in autofluorography, specifically compositions which enhance the detection of radiolabelled substances.
Autoradiography is the process by which an image is generated in a radiation sensitive material, e.g., a photographic emulsion, when exposed to radioactivity. This technique is particularly useful for identification of biological substances such as proteins, amino acids, or nucleic acids. Autoradiography techniques involve radiolabelling the protein or nucleic acid with a radioactive isotope, and then determining the location of the radiolabelled molecule by allowing the radioactive emissions to expose a photoemulsion on the photographic plate or specimen coated with a photographic emulsion. Commonly used radioactive isotopes used for radiolabelling are .sup.3 H, .sup.14 C, .sup.35 S, .sup.125 I and .sup.32 P. The radiolabelled material may be in a chromatograph, an electrophoresis gel, or a tissue specimen.
A major problem in autoradiography is that the low energy and/or radiation level emitted by many of these isotopes when incorporated into a protein or nucleic acid is insufficient to provide clear, rapid exposure of the photographic emulsion. Scintillators or fluors are used in order to achieve this end. Fluors are molecules that absorb the radiation emitted by the radioactive isotopes and emit light The emitted light is more efficient at exposing the photographic emulsion than the emissions from the radioactive decay, leading to quicker and more accurate results.
In some circumstances, a scintillation solvent and/or a secondary fluor is incorporated into the solution in order to improve the efficiency of fluorescence. Scintillation solvents are chemicals which assist in the transfer of the emitted electron radiation from the radioactive isotopes to the fluors, improving the energy transfer and system efficiency. The secondary fluors absorb energy from the primary fluor and emit light at a different wavelength which may improve exposure of the photographic emulsion. The use of the fluor or scintillator system in this image-creating process is called autofluorography.
Autofluorography is useful with a wide variety of separation media and techniques. These include thin layer chromatography, paper chromatography, gel electrophoresis, and column chromatography. The radioactive material may be absorbed or adsorbed to the separation medium. Media which are useful include silica gel, alumina, cellulose, polyamide, polyacrylamide, cross-linked dextran, agarose, and nitrocellulose. These media are normally supported by a plate or other structure.
Despite its broad applicability, there are substantial problems with the use of autofluorography which make it a difficult method to apply. For example, in thin layer chromatography, where the radioactive emitters are at or near the surface of the chromatograph, they interact with the fluor in the surface layer. However, it is difficult to obtain a smooth, even distribution of the fluor even when using a spray technique for application. Uneven application can cause nonreproducible results. Additionally, since the fluor is normally dissolved in a carrier solvent, the radioactive material may migrate with the solvent, causing movement of bands which also can lead to inaccurate results. Further, small crystals of the scintillator which form during the processing may fall off or be moved during handling.
At the other extreme of media thickness, gel electrophoresis and other related media separation techniques use much thicker media, typically greater than 0.1 mm. These thicker media lead to different problems because the material to be detected is normally not located on the surface area but rather is disbursed throughout the media. Since the low energy radiation of the incorporated radioactive isotopes is greatly attenuated by distance, a surface treatment with the fluor is insufficient to visualize a majority of the emitted radiation, leading to inaccuracies. In order to counteract this, the fluor, either alone or in conjunction with an intermediate scintillation solvent and/or secondary fluor, is placed in intimate contact with the radioactive isotope to amplify the signal prior to attenuation. To achieve this contact, it is necessary to uniformly transfer the fluor system throughout the media which insures that there are no differences in results caused by incomplete adsorption of emitted radiation. Generally, this uniform transfer is accomplished by soaking the separation medium in a bath containing the fluor dissolved in a suitable solvent.
There are a number of different scintillation compositions and methods which have been used for enhanced autoradiography to identify materials incorporated into separation media. One system is described by Bonner at al., Eur. J. Biochem. 46(1):83-88 (1974). In this method, the radiolabelled protein is separated by electrophoresis using an aqueous polyacrylamide gel. Separation is followed by soaking the gel in about 20 times its volume of dimethylsufoxide (DMSO) for 30 minutes, and then immersed a second time for 30 minutes in fresh DMSO to displace all the water from the gel. The gel is then soaked in a 20% (w/w) solution of 2,5-diphenyloxazole (PPO) in DMSO to impregnate the gel with the scintillator PPO. The fluor is precipitated in the gel by immersing in water and the gel is dried and exposed to the photographic film.
The Bonner technique has a number of disadvantages, several of which are discussed in the appendix of the Laskey and Mills article in Eur. J. Biochem., 56:335-341 (1975). For example, if agarose gels are used in place of acrylamide, the agarose will dissolve in DMSO. This is counteracted by the substitution of methanol for the DMSO but this substitution is only effective for gels having less than 2% polyacrylamide since gels having higher concentrations of polyacrylamide shrink severely when contacted with methanol. Another disadvantage is the ability of DMSO to penetrate through the skin of anyone handling it. DMSO can carry the dissolved material, some of which may be radioactive and otherwise potentially toxic, with it through the skin. DMSO also imparts a garlic smell to the breath. Other disadvantages reported include the length of time gels must be soaked in the DMSO-fluor solution to obtain complete impregnation, the requirement for high concentrations of PPO, and the time-consuming and waste-generating soakings in DMSO to dehydrate the gel.
Because of these problems, many other methods for incorporating fluors into separation media appear in the literature. Bonner and Stedman, Anal. Biochem., 89: 247-256 (1978), describe methods suitable for thin layer chromatography. These methods use scintillation solvents to increase the absorption ability of 2,5-diphenyloxazole. Although helpful for thin layer chromatography, these methods have serious drawbacks when applied to gel electrophoresis because the gels used generally contain greater than 80%, more often greater than 90%, water. The fluors and solvents used in the Bonner and Stedman methods are not water soluble or water miscible to any appreciable extent so they cannot be used for efficient and uniform transport of the fluors to the interior of the gel. In fact, the fluors tend to precipitate on the surface of the gel. In addition, the suggested organic solvents cause drastic shrinkage of the gel which prevents fluor impregnation and lead to distortion of the gel. In fact, aqueous polyacrylamide or agarose gels cannot be impregnated with 2,5-diphenyloxazole or 2-methylnaphthalene while in the hydrated state using these methods.
U.S. Pat. No. 4,293,436, issued on an application of Fost, describes a different autofluorographic technique which does not require a dehydration step. The aqueous separation medium is impregnated with a combination of a water-soluble or water-miscible lower alkyl carboxylic acid and an alcohol as a swelling inhibitor in which a scintillator fluor has been dissolved. The fluor is precipitated within the gel by aqueous soaking. However, this procedure also has several disadvantages. Proteins must first be fixed in a separate step. Further, there is limited or no reusability of the excess product used for impregnation, which increases costs and generates hazardous wastes. In addition, the combination of carboxylic acid and alcoholic swelling inhibitor is unstable and gradually forms a stable ester. This decreases the suitability of the product for its intended use and limits shelf life.
Another autofluorographic system is described in U.S. Pat. No. 4,522,742, issued on an application of Lee et al. In this technique, the separation medium is impregnated by an aqueous autofluorographic enhancer containing water soluble fluors. This water-based system does not, however, work effectively in very thin gels (&lt;1.0 mm) or gels with less than 5% acrylamide or agarose. No dehydration step is needed since unlike the systems based on organic solvent impregnation and water precipitation, this aqueous system transports fluor into the gel without exchanging the solvent and without precipitating the fluor. However, the gels must be dried prior to film exposure. Water removed by vacuum aspiration contains the water soluble fluor intended for enhancement purposes, thereby requiring high initial fluor concentration. For thin, porous gels in which mechanical entrapment cannot be relied upon until sufficient evaporation has occurred to precipitate the water soluble fluor, enough fluor can be removed by vacuum aspiration to seriously decrease the enhancement process.
Thus, alternative methods for enhancing the detection of radiolabelled materials by means of autoradiography or autofluorography are being sought.